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Identification of Gram Positive Coccus


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Report
Practical work nº 8
Identification of Gram Positive Coccus

General Microbiology (Pratical)

2006

Carlos Alberto Teixeira



Hugo Miguel Sousa

Introoduction

In this practical work a set of operations to allow the differentiation of gram positive bacterias was made.

Executed procedures:


  • Gram Stainning;

  • Meio de Manitol Salt Agar (MSA);

  • Desoxirribonuclease (DNAse) Test;

  • Catalase Test;

  • Coagulase Test;

  • Oxidase Test.

The studied microrganisms were:



  • Staphylococcus aureus;

  • Streptococcus.

Experimental results:

Gram Stainning

Microrganism

Gram

Structure

Staphylococcus aureus

Positive

Coccus, form a grape cluster style structure.

Streptococcus

Positive

Coccus, form rows.



Manitol Salt Agar (MSA) Medium

Microrganism

Growth

Manitol Fermentation

Staphylococcus aureus

There is growth

The medium acidified, therefore there is manitol fermentation.

Streptococcus

There is growth

The medium has acidified, hence there is fermentation of the manitol.



Desoxirribonuclease (DNAse) Test

Microrganism

DNAse

Staphylococcus aureus

Presence of a transparent halo denotes the degradation of the DNA in the medium.

Streptococcus

The precipitation of the DNA within the medium demonstrates that the microrganism did not degrade it.


Catalase Test

Microrganism

Catalase

Staphylococcus aureus

Presence of effervescence, hence positive.

Streptococcus

No alteration so the result is negative.



Coagulase Test

Microrganism

Coagulase

Staphylococcus aureus

Visible coagulum, hence positive result.

Streptococcus

No coagulation so the result is negative.



Oxidase Test

Microrganism

Oxidase

Staphylococcus aureus

No visible alteration, hence negative.

Streptococcus

No visible alteration, therefore negative.

Results discussion and conclusion

By doing the Gram stainning and observing each of the microrganisms at the microscope it is possible to see that they are both Gram positive, due to their purple colour which comes from the lugol + violet cristal complex. Coccus form is also verified in both microrganisms being that the Streptococcus seems to show a more orderly structure, in row, on the opposite, the Staphylococcus aureus cells aglomerate in clusters.


The Manitol Salt Agar, which, being a hypertonic medium (high concentration of NaCl), allows the growth of Gram positive organisms, degrading the Gram negatives. This medium is also used to see the use of manitol by the studied microrganism. With the indicator present in the medium, phenol red, which changed from red (pH=8.4) to yellow (pH=6.8) it was possible to understand that the medium acidified, which means that the sugar was fermented.
The DNAse test was done to check if any of the microrganisms contained the Desoxirribonuclease enzyme. It was verified with the addition of Chlorine Acid (HCl) with a concentration of 1 Molar. After this procedure, it was possible to verify that around Staphylococcus aureus there was a transparent hal, which indicates that the DNA present in the medium was degradated by the microrganism. Around the Streptococcus a rash was found within the whole plate, which demonstrates that the microrganism does not detain the enzyme.

Using a slide with a few drops of hydrogen peroxide (H2O2) it was possible to determine whether the microrganisms in study possessed the catalase enzyme. This enzyme decomposes the solution with an oxidoreductase in compliance with the equations:

H2O2 + Fe-E → H2O + O=Fe-E

H2O2 + O=Fe-E → H2O + Fe-E + O2

being that Fe-E indicates the iron present in the heme group connected to the catalase enzyme.

Through this reaction, which unleashes an effervescence, it is possible to determine, visually, if the microrganism detains the enzyme. Staphylococcus aureus was able to produce it, which implies that it is catalase positive, on the other hand the Streptococcus wasn’t able to change the H2O2 solution.

Coagulase is an enzyme which provokes a goagulation of the blood plasma, in the present case, from rabbit, due to the deneutralizing of its fibrine.

In case of a coagulum if formed, as in the case of the one where the Staphylococcus aureus was innoculated, there is the indication that the microrganism detains the enzyme, and is demonstrated that it is extremely dangerous if it enters the blood stream as it can create disseminated intravascular coagulums. In the case of the tube innoculated with the Streptococcus the result was negative.


The intracellular oxidase enzyme is the responsible for the activation, in the presence of a cytochrome oxidase system, of the cytochrome oxidation reduced by the molecular oxygen, which, in turn, will act as a final receptor of the electrons in the final stage of the eletron transference. The molecular oxygen is then transformed into water. During the process, it helps to establish a electro-chemical potential that another enzyme (the ATP sintase) will use to sintetize the ATP molecule. All this process is known as chemiosmotic.

The indophenol is oxidated in the presence of microrganisms that produce the oxidase in the presence of oxygen, the cytochrome C oxidase and the oxidase reagent phenilendiamin, producing a purple composite.



It is concluded with this test that both the microrganisms in study do not detain the intracellular oxidase enzyme since when they were placed in cantact with the reagents strip the result was negative.

Bibliography


  • http://en.wikipedia.org/wiki/Staphylococcus_aureus

  • http://en.wikipedia.org/wiki/Streptococcus

  • http://en.wikipedia.org/wiki/Cytochrome_c_oxidase

  • http://en.wikipedia.org/wiki/Coagulase

  • http://en.wikipedia.org/wiki/Catalase

  • http://en.wikipedia.org/wiki/Chemiosmotic_potential

  • http://academic.scranton.edu/faculty/CANNM1/biochemistry/biochemistrymoduleport.html

  • General Microbiologia classes 2005-2006



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